The invention herein concerns the labeling of cells, usually in the bloodstream, which are capable of phagocytosis. More specifically, the invention provides a method for supplying a label to the cells by taking advantage of their phagocytic properties with respect to suitably labeled micellular particles such as vesicles which have surface amino groups. In the method of the invention, the label is carried to and entrapped in the phagocytic cell by subjecting these micellular particles to endocytosis.
There is an extensive technology relating to the problem of labeling specific types of cells so that they can be traced and located within an organism. A recent survey of the state of the art concerning radioactive labels, particularly, the gamma-emitter indium-111 (In-111), is summarized in Indium-111 Labeled Neutrophils, Platelets and Lymphocytes, Proceedings of the Yale Symposium, New York City, N.Y., Sept. 14 and 15, 1979, Trivirum Publishing Company, N.Y. (1980).
One approach that has frequently been used for labeling cells is that of isolating the cells desired to be labeled, and then supplying the labeling material only to these cells. This approach, of course, has the inherent disadvantage of requiring clean separation of the desired cells from other components associated with them, which, at best, constitutes an additional step. Even the relatively straightforward separation of blood into red blood cells and plasma is a troublesome step, if this technique is to be used for in vivo diagnosis, because the performance of this step mandates a longer residence time for the cells outside the body. Other separations are even more subtle. For example, it is particularly difficult to separate blood monocytes from neutrophils as well as from lymphocytes (ibid, page 5). The technique in common use today for introducing In-111 to cells, which employs indium complexed with 8-hydroxy-quinoline (oxine), is non-specific with respect to these cells and requires prior isolation if one cell type is to be targeted.
An alternative approach has been to use specific labeling agents which are known to prefer a particular type of cell. An example is the use of .sup.32 p diisopropylfluorophosphate (DF.sup.32 p) for the specific binding of neutrophils in preference to other blood components (ibid, page 1). However, since this reagent is a beta-emitter, a tracing method which uses this label is inherently less sensitive than any method which uses indium-111. Other commonly used labels include chromium-51, gallium-67 and radioactive isotopes of iodine. All of these labels suffer from the non-specificity exhibited by their known chemically combined forms, as well as from other problems.
The following references provide background for the invention and are incorporated into the herein application by reference.
1. Burleson, R. L., Johnson, M. C. & Head, H. (1973) Ann. Surg., 178, 446.Scintigraphic demonstration of experimental abscesses with intravenous .sup.67 Ga citrate and .sup.67 Ga labeled blood leukocytes. PA1 2. Burleson, R. L., Johnson, M. C. & Head, H. (1974) J. Nucl. Med., 15, 98. In vitro and in vivo labeling of rabbit blood leukocytes with .sup.67 Ga citrate. PA1 3. Forstrom, L., L. Gomez, B. Weiblen, D. Hoogland, J. McCullough, & M. Loken (1978) J. Nucl. Med., 19, 672. Clinical use of indium-111 oxine labeled leukocytes in the detection of inflamation or abscess. PA1 4. Zakhireh, B., M. L. Thakur, H. L. Malech, M. S. Cohen, A. Gottschalk, & R. K. Root (1979) J. Nucl. Med., 20, 741. Indium-111-labeled human polymorphonuclear leukocytes: Viability, random migration, chemotaxis, bactericidal capacity, and ultrastructure. PA1 5. Dutcher, J. P., C. A. Schiffer & G. S. Johnson (1981) N. Eng. J. Med., 304, 586. Rapid migration of indium-111-labeled granulocytes to sites of infection. PA1 6. Alavi, J. B., M. M. Staum & A. Alavi (1980) "In-111 for granulocyte labeling in neutropenic patients" in Indium-111 Labeled Neutrophils, Platelets and Lymphocytes, eds. M. L. Thakur & A. Gottschalk (Trivirum Publishing Co., New York) pp. 41-50. PA1 7. Forstrom, L. A., B. J. Weiblen, L. Gomez, N. L. Ascher, D. R. Hoogland, M. K. Loken, & J. McCullough (1980) "Indium-111-oxine-labeled leukocytes in the diagnosis of occult inflammatory disease" in Indium-111 Labeled Neutrophils, Platelets and Lymphocytes, eds. M. L. Thakur & A. Gottschalk (Trivirum Publishing Co., New York) pp. 123-140. PA1 8. Goodwin, D. A., P. W. Doherty & I. R. McDougall (1980) "Clinical use of In-111-labeled white cells: An analysis of 312 cases" in Indium-111 Labeled Neutrophils, Platelets and Lymphocytes, eds. M. L. Thakur & A. Gottschalk (Trivirum Publishing Co., New York) pp. 131-150. PA1 9. Thakur, M. L., J. P. Lavender & R. N. Arnot (1977) J. Nucl. Med., 18, 1014. Indium-111-labeled autologous leukocytes in man. PA1 10. Mauk, M. R., R. C. Gamble & J. D. Baldeschwieler (1980) Science, 207, 309. Vesicle targeting: Time release and specificity for the leukocytes system in by subcutaneous injection. PA1 11. Mauk, M. R., R. C. Gamble & J. D. Baldeschwieler (1980) Proc. Natl. Acad. Sci. USA, 77, 4430. Targeting of lipid vesicles: Specificity of carbohydrate receptor analogues for leukocytes in mice. PA1 12. Wu, P. S., G. W. Tin & J. D. Baldeschwieler (1981) Proc. Natl. Acad. Sci. USA, 78, 2033. Phagocytosis of carbohydrate-modified phospholipid vesicle by macrophage.
Hereinafter, citation to these references shall be done by inserting the number of a cited reference within a parenthesis.
The tracing of phagocytic cells is of particular importance because they tend to accumulate at the sites of infections and abscesses. At the present time, a significant number of the numerous abdominal surgeries performed annually to repair wounds and for corrective purposes result in occult infections which require intensive care. Also, the formation of microabscesses at the interface of an organ transplant is an indicator of the first stages of tissue rejection. It is known, of course, that certain white blood cells accumulate at these infection sites, and attempts have been made to use leukocyte labeling to permit detection of these accumulations (1, 2). Since the known techniques are non-specific, use of the radiolabeling techniques discussed above to detect these occult infections necessitates a prior separation of leukocytes by isolating blood from the patient, separating the leukocyte rich plasma from the red blood cells by centrifugation, incubating the plasma with, for example, In-111 oxine, and then reinjecting the labeled plasma into the patient. Gamma imaging of the patient is used to locate aggregations of the labeled leukocytes which are attracted to the site of an infection (3, 4, 5).
However, this approach has not solved the problem of non-specificity which has been experienced with other reagents because of background radiation due to an accumulation of the In-111 in undesired locations (6, 7, 8). This accumulation occurs either because the In-111 was not exclusively attached to the leukocyte in the first place, (but also, for example, to the red blood cells) or because a portion of the In-111 becomes deposited in the liver, spleen and other organs by virtue of damage to carrier leukocytes occurring in the separation or labeling processes, or both. Further, the dumping of In-111 onto non-targeted sites by damaged leukocytes results in retention of radioactivity at these locations which has the consequence of increasing the patient's exposure to radiation (9).
Other difficulties have been found. The diminution of the phagocytic and chemotactic abilities of the leukocytes which have been exposed to the reagents used for labeling has also reduced the effectiveness of the method (4, 5). Further, there is the general disadvantage of a 2 to 3 hour residence time for the leukocytes outside the body of the patient because of the necessity for plasma separation.
The method of the present invention overcomes these difficulties by providing a more effective technique for specifically labeling leukocytes, either while outside the patient's body or by injection, which uses micellular particles such as vesicles as a carrier for a label. Significantly, the method does not require the separation of blood into its various cell types even though partial or complete separation is not inconsistent with the method of the invention and may have advantages in certain applications.
The invention takes advantage of, and extends, the observed property of incorporating an extended amine on a micelle's surface to cause aggregation of polymorphonuclear leukocytes when injected subcutaneously (10, 11) and to enhance phagocytosis by peritoneal macrophages in vitro (12).
In the process of the invention, rather than employing oxine, the In-111 (or other label) is enclosed in, or otherwise bound to the micellular particles which can be phagocytosed by the leukocytes but which do not bond or otherwise become fixed to other cells, thereby permiting and encouraging the incorporation of the label into the phagocytic cells, which, in turn seek out and accumulate at sites of occult infection. Vesicles are particularly preferred as the micellular particle.